Ultrastructural analysis of different skeletal cell types in mucopolysaccharidosis dogs at the onset of postnatal growth.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.

Insight into bone-derived biological apatite: ultrastructure and effect of thermal treatment.

OBJECTIVES: This study aims at examining the ultrastructure of bone-derived biological apatite (BAp) from a series of small vertebrates and the effect of thermal treatment on its physiochemical properties. MATERIALS AND METHODS: Femurs/fin rays and vertebral bodies of 5 kinds of small vertebrates were firstly analyzed with X-ray microtomography. Subsequently, BAp was obtained with thermal treatment and low power plasma ashing, respectively. The properties of BAp, including morphology, functional groups, and crystal characteristics were then analyzed. RESULTS: The bones of grouper and hairtail were mainly composed of condensed bone. Spongy bone showed different distribution in the bones from frog, rat, and pigeon. No significant difference was found in bone mineral density of condensed bone and trabecular thickness of spongy bone. Only platelet-like crystals were observed for BAp obtained by plasma ashing, while rod-like and irregular crystals were both harvested from the bones treated by sintering. A much higher degree of crystallinity and larger crystal size but a lower content of carbonate were detected in the latter. CONCLUSION: Platelet-like BAp is the common inorganic component of vertebrate bones. BAp distributing in condensed and spongy bone may exhibit differing thermal reactivity. Thermal treatment may alter BAp's in vivo structure and composition.

Morphological differences in adolescent idiopathic scoliosis: a histological and ultrastructural investigation.

STUDY DESIGN: Histological and ultrastructural evaluation of cell morphologies at the concave and convex side of apical intervertebral discs (IVD) of adolescent idiopathic scoliosis (AIS). OBJECTIVE: To determine changes in cell morphology, viability, and cell death after asymmetric disc loading in AIS and to compare the findings with the tilt angles. SUMMARY OF BACKGROUND DATA: The reaction of cells to loading stimuli in the IVD seems to be specific. Although dynamic loads are more beneficial to the disc cells and maintain the matrix biosynthesis, static compressive loads suppress gene expression. METHODS: Apical IVDs (Th8-Th9 to L1-L2) from 10 patients with AIS were studied histologically (including TUNEL [TdT-mediated dUTP-biotin nick end labeling] staining to identify disc cell death by apoptosis) and ultrastructurally for matrix evaluations and to quantify healthy, balloon, chondroptotic, apoptotic, and necrotic cells on the concave and convex sides. Patients' spines were classified according to the Lenke classification. Degeneration was assessed according to the Pfirrmann grading system. Two groups were established; group 1 (G1) with a tilt of 5° to 9° and group 2 (G2) with a tilt of 10° to 19°. RESULTS: Balloon cells were found in significantly higher numbers at the concave side (G1-annulus fibrosus [AF]: mean 16%), with almost none found at the convex side. Mean numbers of healthy cells did not show differences comparing both sides. Significantly higher numbers of healthy cells were found with increasing tilt angle at the concave side. Necrosis (mean, 47%) increased toward the center of the disc but did not differ between the sides of the IVDs. The fibrils found in the outer AF on the convex side were 30% thinner. CONCLUSION: This study was able to show significant differences in cell morphologies in the AF on both sides and in correlation to the different tilt angles. The type and magnitude of load seem to influence disc cells. Further studies are required to provide more information on disc and cell changes in scoliosis.

Spirocerca lupi-associated vertebral changes: a radiologic-pathologic study.

Spirocerca lupi causes a caudal esophageal mass in dogs which may be accompanied by aortic changes and caudal thoracic spondylitis. Previous literature hypothesized that the spondylitis was caused by either aberrant larval migration or was secondary to the inflammation caused by the aortic migration. The current study aimed to evaluate these hypotheses. Ten dogs of various breeds and ages with radiographic evidence of spondylitis, which were necropsied, had the affected vertebrae removed and prepared for light and transmission electron microscopy examination. Transverse and sagittal sections of the ventral vertebrae were taken from 27 spondylitis and 8 spondylosis deformans lesions as well as from 8 normal vertebrae. Early spondylitis changes were characterized by periosteal woven new bone covered by hyperplastic periosteum with some involvement of the ventral longitudinal ligament. More mature lesions were characterized by nodules of denser trabecular bone and cartilage, also covered by hyperplastic periosteum and involved the ventral longitudinal ligament. It was difficult to distinguish the spondylitis and spondylosis deformans new bone. Inflammation was seen in five spondylitis cases (edema, lymphocytes, plasma cells, eosinophils and fibrin fibers). Spirocerca eggs were seen in one histologic section. This study shows that inflammation is mild and inconsistent in spirocercosis-induced spondylitis and that aberrant migration of the larvae or adults did not appear to be a predominant cause. Inflammatory mediators or osteoproliferative growth factors, which may be related to the primary esophageal lesion or to the worm itself, could be involved. This requires further investigation.

The effect of intravertebral heterogeneity in microstructure on vertebral strength and failure patterns.

UNLABELLED: The goal of this study was to determine the influence of intravertebral heterogeneity in microstructure on vertebral failure. Results show that noninvasive assessments of the intravertebral heterogeneity in density improve predictions of vertebral strength and that local variations in microstructure are associated with locations of failure in the vertebral body. INTRODUCTION: The overall goal of this study was to determine the influence of intravertebral heterogeneity in microstructure on vertebral failure. METHODS: Trabecular density and microarchitecture were quantified for 32 thoracic vertebrae using micro-computed tomography (µCT)-based analyses of 4.81 mm, contiguous cubes throughout the centrum. Intravertebral heterogeneity in density was defined as the interquartile range and quartile coefficient of variation of the cube densities. The vertebrae were compressed to failure to measure stiffness, strength, and toughness. Pre- and post-compression µCT images were analyzed using digital volume correlation to quantify failure patterns in the vertebrae, as defined by the distributions of residual strain. RESULTS: Failure patterns consisted of large deformations in the midtransverse plane with concomitant endplate biconcavity and were linked to the intravertebral distribution of bone tissue. Low values of connectivity density and trabecular number, and high values of trabecular separation, were associated with high strains. However, local microstructural properties were not the sole determinants of failure. For instance, the midtransverse plane experienced the highest strain (p < 0.008) yet had the highest density, lowest structure model index, and lowest anisotropy (p < 0.013). Accounting for the intravertebral heterogeneity in density improved predictions of strength and stiffness as compared to predictions based only on mean density (strength: R(2) = 0.75 vs. 0.61, p < 0.001; stiffness: R(2) = 0.44 vs. 0.26, p = 0.001). CONCLUSIONS: Local variations in microstructure are associated with failure patterns in the vertebra. Noninvasive assessments of the intravertebral heterogeneity in density--which are feasible in clinical settings--can improve predictions of vertebral strength and stiffness.

What is the best way to optimize thoracic kyphosis correction? A micro-CT and biomechanical analysis of pedicle morphology and screw failure.

STUDY DESIGN: A human cadaveric biomechanical analysis. OBJECTIVE: The purpose of this study was to evaluate the bone density/trabecular width of the thoracic pedicle and correlate that with its resistance against compressive loading used during correction maneuvers in the thoracic spine (i.e., cantilever bending). SUMMARY OF BACKGROUND DATA: As surgeons perform cantilever correction maneuvers in the spine, it is common to have pedicle screws pullout or displace while placing corrective forces on the construct. Currently, surgeons either compress against the cephalad aspect of the pedicle or vice versa. We set out to establish which aspect of the pedicle was the most dense and to determine the optimal direction for screw compression during kyphosis/deformity correction. METHODS: Fifteen fresh-frozen cadaveric vertebrae (n = 15) were examined by micro-computed tomography to determine percent bone volume/total volume (%BV/TV) within the cephalad and caudad aspects of the pedicle. Specimens were sectioned in the sagittal plane. Pedicles were instrumented according to the straightforward trajectory on both sides. Specimens were then mounted and loading to failure was performed perpendicular to the screw axis (either the cephalad or the caudad aspect of the pedicle). RESULTS: Mean failure when loading against the caudad aspect of the pedicle was statistically, significantly greater (454.5 ± 241.3 N vs. 334.79 1 ± 158.435 N) than for the cephalad pedicle (P < 0.001). In concordance with failure data, more trabecular and cortical bones were observed within the caudad half of the pedicle compared with the cephalad half (P < 0.001). CONCLUSION: Our results suggest that the caudad half of the pedicle is denser and withstands higher forces compared with the cephalad aspect. In turn, the incidence of intraoperative screw loosening and/or pedicle fracture may be reduced if the compressive forces (cantilever bending during deformity correction) placed upon the construct are applied against the caudad portion of the pedicle.

Electron-microscopic imaging of endothoracic fascia in the thoracic paravertebral space in rats.

BACKGROUND AND OBJECTIVES: Anesthesia and analgesia with paravertebral block are reportedly variable. Existence of an endothoracic fascia has been proposed as one of the possible mechanisms leading to variability. We undertook an electron-microscopy imaging study to investigate the endothoracic fascia in the thoracic paravertebral space (TPS) in rats. METHODS: Male Wistar rats were studied in accordance with the principles of laboratory animal care. After the rats were euthanized in a CO2 chamber, the thoracic paravertebral tissues were removed en bloc and cut into consecutive transverse sections of approximately 3 mm. Stereomicroscopy and electron-microscopy assessments were performed by 2 independent observers. RESULTS: The endothoracic fascia was consistently identified in all specimens. The fascia was located between the parietal pleura and the innermost intercostal muscles or ribs. Its thickness ranged from 15 to 27 µm (mean, 20 ± 3 µm). The endothoracic fascia divided the TPS in 2 compartments: one, extrapleural and anterolateral (EPC); another, subendothoracic and posteromedial (SETC). The spinal nerves with their ganglia were found within SETC, whereas the sympathetic ganglia were consistently located within the EPC. CONCLUSIONS: The endothoracic fascia in rats appears to divide the TPS into EPC and SETC. These anatomic characteristics may have implications in thoracic paravertebral blockade.

Morphologic study of nerve root and types of needle used in transforaminal injections.

BACKGROUND: The bevel type and location of the distal orifice of the needle may have relevance for potential complications occurring during transforaminal epidural injection. METHODS: We examined by scanning electron microscopy the structural aspects of spinal nerve root cuffs of 3 human cadavers, and 3 needle types used in transforaminal injections: 22-gauge Quincke spinal needles, 22-gauge blunt nerve block needles, and 20-gauge radiofrequency blunt needles. We made punctures in vitro in the spinal nerve root cuffs, and we studied the structures affected. RESULTS: There is fat tissue within the nerve root with irregular distribution. In needles with a round tip, the distal orifice maintained an extraneural location after puncture of the nerve root cuff. The length of the needle required to introduce the distal orifice completely inside the nerve root cuff was variable, depending on the type: shortest for a Quincke needle (1.8 mm), 4.1 mm for the Epimed, and longest for radiofrequency needles (5.7 mm). CONCLUSIONS: The layer of fat around nerve roots may prevent the contact of the needle tip with axons. The sharp needle tip entered the nerve root cuff more easily than the blunt tip in the cadaveric nerve root samples, measured in a qualitative manner. There is a need for clinical studies to ascertain if blunt needles may be safer than sharp needles for transforaminal injections.

Micro-computed tomography study of the subchondral bone of the vertebral endplates in a porcine model: correlations with histomorphometric parameters.

PURPOSE: Subchondral bone (SCB) of the vertebral endplates (VEP) is the principal site of changes in vertebral trabecular microarchitecture secondary to intervertebral disc degeneration. However, the microstructure of this region has not yet been clearly characterized. METHODS: One thoracic and one lumbar vertebral unit (vertebra-disc-vertebra) was removed in nine pigs aged 4 months. Three samples (one central and two laterals) were taken from each VEP. Micro-CT examination and histomorphometric measurements of the subchondral trabecular bone of the VEP were carried out. Correlations between micro-CT and histological parameters were sought. RESULTS: Trabecular network was significantly denser [increased bone volume fraction (BV/TV) and trabecular number (Tb.N), decreased intertrabecular separation (Tb.Sp)] in the cranial endplates of the vertebral units. It was also significantly denser and less well organized [increased degree of anisotropy (DA)] in the centre of the VEP. The thickness of the cartilage endplate (CEP), SCB and growth cartilage were significantly lower in the centre of the VEP. There was a significant negative correlation between BV/TV, Tb.N and DA with the thicknesses of the CEP and SCB whereas Tb.Sp was positively correlated with these two parameters. CONCLUSION: We observed densification of the trabecular network in the centre of the VEP overlying the nucleus pulposus, partly related to thinner hyaline cartilage. Densification is associated with more anisotropic architecture that could cause lower mechanical strength in this area. This study provides new information on the microarchitecture of the SCB of the VEP which will make it possible to validate future models.

Increased calcium content and inhomogeneity of mineralization render bone toughness in osteoporosis: mineralization, morphology and biomechanics of human single trabeculae.

The differentiation and degree of the effects of mineral content and/or morphology on bone quality remain, to a large extent, unanswered due to several microarchitectural particularities in spatial measuring fields (e.g., force transfer, trajectories, microcalli). Therefore, as the smallest basic component of cancellous bone, we focused on single trabeculae to investigate the effects of mineralization and structure, both independently and in superposition. Transiliac Bordier bone cores and T12 vertebrae were obtained from 20 females at autopsy for specimen preparation, enabling radiographical analyses, histomorphometry, Bone Mineral Density Distribution (BMDD) analyses, and trabecular singularization to be performed. Evaluated contact X-rays and histomorphometric limits from cases with osteoporotic vertebral fractures generated two subdivisions, osteoporotic (n=12, Ø 78 years) and non-osteoporotic (n=8, Ø 49 years) cases, based on fracture appearance and bone volume (BV/TV). Measurements of trabecular number (Tb.N.), trabecular separation (Tb.Sp.), trabecular thickness (Tb.Th.), trabecular bone pattern factor (TBPf) and eroded surface (ES/BS) were carried out to provide detailed structural properties of the investigated groups. The mechanical properties of 400 rod-like single vertebral trabeculae, assessed by three-point bending, were matched with mineral properties as quantified by BMDD analyses of cross-sectioned rod-like and plate-like trabeculae, both in superposition and independently. Non-osteoporotic iliac crests and vertebrae displayed linear dependency on structure parameters, whereas osteoporotic compartments proved to be non-correlated with bone structure. Independent of trabecular thickness, osteoporotic rod-like trabeculae showed decreases in Young's modulus, fracture load, yield strength, ultimate stress, work to failure and bending stiffness, along with significantly increased mean calcium content and calcium width. Non-osteoporotic trabeculae showed biomechanically beneficial properties due to a homogeneous mineralization configuration, whereas osteoporotic trabeculae predominantly demonstrated various mineralized bone packets, eroded surfaces, highly mineralized cement lines and microcracks. The Young's moduli of single trabeculae exhibited significantly negative linear correlations with trabecular thickness. Because of increased, but inhomogeneously distributed, calcium content, osteoporotic trabeculae may be subject to shear stresses that render bone fragile beyond structure impairment due to cracks and lacunae.